Effects of ouabain on chloride movements across the seawater eel intestine

Summary Simultaneous measurements of transepithelial potential difference (PD) and net water flux were made in the stripped intestine of seawater eels, and the effects of ouabain on these two parameters were examined in normal Ringer solution or under a chloride concentration gradient. Ouabain reduced the serosa-negative PD and the net water flux in normal Ringer solution with a linear relationship between the PD and the net water flux. Removal of K⁺ from the Ringer solution on both serosal and mucosal sides also reduced the PD and the net water flux to approximately zero. On the other hand, blocking the Na⁺–K⁺ pump by ouabain, K⁺-free or Na⁺-free Ringer solution increased the diffusion potential for Cl^–. Inhibition of Cl^– transport and increment in Cl^– permeability by ouabain occurred almost simultaneously. It is likely, therefore, that Cl^– transport as well as Cl^– permeability is dependent on Na⁺–K⁺ pump activity. A possible mechanism of dependence of Cl^– transport on the Na⁺–K⁺ pump is discussed in relation to the increment in Cl^– permeability.     

ESR studies of light-dependent volume changes in cell envelope vesicles from Halobacterium halobium

Volume changes in illuminated cell envelope vesicles, prepared from various Halobacterium halobium strains, were measured with an ESR method. We demonstrated light-dependent swelling of vesicles which contained halorhodopsin (an inward-directed light-driven chloride pump), and shrinking of vesicles which contained bacteriorhodopsin (an outward-directed light-driven proton pump coupled to a proton/sodium antiporter). The swelling of the halorhodopsin vesicles was not inhibited by uncouplers or gramicidin, but the shrinking of the bacteriorhodopsin-vesicles was abolished by these ionophores. These findings confirm earlier models for ion circulation in these systems. Vesicles from strains which contained both pigments showed relatively small net volume changes upon illumination. A scheme of ionic transport in H. halobium cells is suggested, in which the inward movement of K⁺ exceeds the outward movement of Na⁺, and the difference equals the Cl⁻ uptake, so as to provide the net gain of KCl necessary for volume increases during cell growth.     

Properties of the Isolated Intact Chloroplast at Cytoplasmic K Concentrations : I. Light-Induced Cation Uptake into Intact Chloroplasts is Driven by an Electrical Potential Difference

Photosynthesis, stroma-pH, and internal K⁺ and Cl⁻ concentrations of isolated intact chloroplasts from Spinacia oleracea, as well as ion (K⁺, H⁺, Cl⁻) movements across the envelope, were measured over a wide range of external KCl concentrations (1-100 millimolar).

Isolated intact chloroplasts are a Donnan system which accumulates cations (K⁺ or added Tetraphenylphosphonium⁺) and excludes anions (Cl⁻) at low ionic strength of the medium. The internally negative dark potential becomes still more negative in the light as estimated by Tetraphenylphosphonium⁺ distribution. At 100 millimolar external KCl, potentials both in the light and in the dark and also the light-induced uptake of K⁺ or Na⁺ and the release of protons all become very small. Light-induced K⁺ uptake is not abolished by valinomycin suggesting that the K⁺ uptake is not primarily active. Intact chloroplasts contain higher K⁺ concentrations (112-157 millimolar) than chloroplasts isolated in standard media. Photosynthetic activity of intact chloroplasts is higher at 100 millimolar external KCl than at 5 to 25 millimolar. The pH optimum of CO₂ fixation at high K⁺ concentrations is broadened towards low pH values. This can be correlated with the observation that high external KCl concentrations at a constant pH of the suspending medium produce an increase of stroma-pH both in the light and in the dark. These results demonstrate a requirement of high external concentrations of monovalent cations for CO₂ fixation in intact chloroplasts.

     

Relationship between coupled Na+−K+−Cl− transport and water absorption across the seawater eel intestine

Summary Simultaneous measurements of net ion and water fluxes were made in the stripped intestine of the seawater eel, and the relationship between Na⁺, K⁺, Cl^– and water transport were examined in the presence of mucosal KCl and serosal NaCl Ringer (standard condition). When Cl^– was removed from both sides of the intestine, net K⁺ flux from mucosa to serosa was reduced, accompanied by complete blockage of water absorption. Since it has been shown that net Cl^– and water fluxes depend on K⁺ transport under the standard condition (Ando 1983), the interdependence of K⁺ and Cl^– transport suggests the existence of a coupled KCl transport system, while the parallelism between the net Cl^– and water fluxes suggests that water absorption is linked to the coupled KCl transport. The coupled KCl and water transport were inhibited by treatment with ouabain or with Na⁺-free Ringer solutions, suggesting the existence of a Na⁺-dependent KCl transport system and linkage of water absorption to the coupled Na⁺–K⁺–Cl^– transport. Since ouabain blocked the active Na⁺–K⁺–Cl^– transport almost completely, the permeability coefficients for K⁺ and Na⁺ through the paracellular shunt pathway were estimated as P_K=0.076 and P_Na=0.058 cm/h, and P_Cl was calculated as 0.005 cm/h. Although Na⁺-independent K⁺ and Cl^tt- fluxes were observed again in the present study, these fluxes were not inhibited by CN^–, ouabain or diuretics, and evoked even after blocking the Na⁺–K⁺–Cl^– transport completely with ouabain. These results indicate that the Na⁺-independent K⁺ and Cl^– fluxes are distinct from the active Na⁺–K⁺–Cl^– transport and are not themselves active.     

Über den lichtgeförderten Influx von Ionen in Blättern von Elodea densa. Vergleich der Influxe von K+- und Cl--Ionen

Summary The light-dependent influxes of K⁺ and Cl^- in detached leaves of Elodea densa were measured using ³⁶Cl^- and ⁴²K⁺ or ⁸⁶Rb⁺ as tracers.The K⁺ and Cl^- influxes were enhanced by light and also in the dark after a preillumination. The rate of influx decayed in the dark according to a first order reaction with a half-time of 25 or 27 sec.DCMU inhibits the light-dependent K⁺ influx more severely in the presence of CO₂ than in its absence in an atmosphere of N₂ containing a trace of oxygen. This is similar to the effect of DCMU on the Cl^- influx. CCCP¹, atebrin (quinacrine) and Dio-9 all affect the influx of K⁺ and Cl^- in a comparable way. CCCP exerts the strongest effect at low light intensities; atebrin and Dio-9 inhibit strongly even at high intensities when the ion influxes are light-saturated. The influence of these two inhibitors in attributed to an effect at the cellular membranes in addition to an effect on photophosphorylation. The effect of CCCP is ascribed to uncoupling of photophosphorylation, as photosynthesis is inhibited by about the same concentration as is ion influx.In far-red light the relative quantum yields of K⁺ and Cl^- influx drop to a similar degree as does the quantum yield of photosynthesis. Estimated values of the quantum requirement of ion influx are given. The quantum requirement in air is higher than in an atmosphere of N₂. It is a function of ion concentration and is lower at higher external concentrations.The results indicate that the K⁺ and Cl^- influxes are partially coupled. The linkage of the ion influxes with the energy sources in the light and a possible contribution of a pseudocyclic photophosphorylation are discussed.     

Ion channels in the membrane ofChara inflata

Summary Voltage-clamped steps in the electric potential difference (PD) across the membrane in cells of the green alga,Chara inflata, cause voltage- and time-dependent current flows, interpreted to arise from opening and closing of various types of ion channel in the membrane. With cells in the light, these channels are normally closed, and the resting PD is probably determined by the operation of an H⁺ efflux pump. Positive steps in PD from the resting level often caused the opening of K⁺ channels with sigmoid kinetics. The channels began to show opening when the PD–120 mV for an external concentration of K⁺ of 1.0mm. Return of the PD to the resting level caused closing of the channels with complex kinetics. Various treatments of the cell could cause these K⁺ channels to open, and remain open continuously, with the PD then lying closer to the Nernst PD for K⁺. The K⁺ channels have been identified by the blocking effects of TEA⁺. Another group of channels, probably Cl^– and Ca²⁺ associated with the action potential open when the PD is stepped to values less negative than –50 mV. Negative steps from the resting PD cause the slow opening, with a time course of seconds, of yet another type of channel, probably Cl^–.     

Measurements of K+-driven Cl− uptake in thylakoids: Inhibition of uptake by antibodies raised to the major polypeptides of the Cl−-efflux active particle(s)

The mechanism of a K⁺-driven Cl^– accumulation against a concentration gradient was investigated by flow dialysis after addition of K⁺-Hepes. Non-specific chloride binding, measured in the presence of choline-Hepes, accounted for approximately 50% of the observed uptake in this system. The K⁺-Hepesdriven Cl^– uptake was inhibited by poly-l-lysine and by two antibodies raised to the major polypeptides of the Cl^–-efflux active particle. Poly-l-lysine had no effect on Cl^– binding estimated with choline-Hepes.     

Chloride transport and the membrane potential in the marine alga,Halicystis parvula

Summary The relationship between the rate of Cl^– transport and the electrical properties ofHalicystis parvula was investigated. Three metabolic inhibitors-darkness, cyanide (2mm), and low temperature (4°C)-all rapidly and reversibly reduce both the short circuit current (SCC), which is a measure of net Cl^– transport, and the vacuole electrical potential (PD). Plotting thePD vs. SCC for inhibited cells yields a linear regression with ay-intercept of zero. ThePD is also greatly reduced when the [Cl^–] of the external medium is lowered. Raising the external [K⁺] produces an appreciable, but less than Nernstian, depolarization, while increasing the external [H⁺] tenfold has no net effect on thePD. Decreasing the external [Na⁺] by tenfold produces only a slight depolarization. Thus, the outer plasma membrane appears to be moderately selective for K⁺ over Na⁺ or H⁺. The effects of ion substitutions in the vacuolar perfusing solutions on thePD reveal that the vacuolar membrane does not discriminate electrically between Cl^– and the much larger anions, isethionate and benzenesulfonate, or between Na⁺ and K⁺. The data suggest that in internally perfused cells ofH. parvula generation of thePD of –50 to –60 mV by a transport system involving only electroneutral pumps is unlikely and that most of thisPD is generated by an electrogenic Cl^– pump.     

Studies on the mechanism of K+ transport in yeast.

The effect of uncouplers and diffusible acids on K⁺ transport was studied in yeast.Although the K⁺ transport system seems to depend on ATP to function, the effects of uncouplers are not due primarily to its action on the energy conserving systems of the cell.Other uncouplers with different structures to that of DNP showed also an inhibitory effect on K⁺ transport, which agrees with their reported ability to conduct protons through membranes.Uncouplers, besides inhibiting K⁺ uptake, produce an efflux of this cation; however, the rate of efflux produced is quantitatively important only when the cells have previously taken up the cation; there seems to exist a mechanism which prevents the loss of cations by yeast.In the absence of substrate, at pH 8.5, with 0.5 m KCl, TCS produces the efflux of H⁺, and when ⁸⁶Rb⁺ was used as a substitute for K⁺, an increase of the entrance of the cation could be detected in the presence of the uncoupler. It seems that the effect of the uncoupler depends on the direction of the combined H⁺ and K⁺ gradients, or the electrochemical potential of the cell.As reported by other authors, weak diffusible acids increase the uptake of K⁺ by yeast, and this effect is not due to changes in the metabolism, but to the magnitude of the entrance of the molecules to the yeast cell.It was found that the efflux of the acids (H₂CO₃), on the other hand, can produce an efflux of K⁺, which means that anions are important not only for the entrance of the cations, but for its permanence within the cell as well.The data seem to be in agreement with the hypothesis of the existence of a proton pump, responsible for the creation of an electrochemical potential, involved in K⁺ transport. At low pH, this pump seems to be activated by the transport of K⁺ into the cell.     

Na+-coupled Cl− transport in the gastric mucosa of the guinea pig

The Cl^–/HCO ₃ ^– exchange mechanism usually postulated to occur in gastric mucosa cannot account for the Na⁺-dependent electrogenic serosal to mucosal Cl^– transport often observed. It was recently suggested that an additional Cl^– transport mechanism driven by the Na⁺ electrochemical potential gradient may be present on the serosal side of the tissue. To verify this, we have studied Cl^– transport in guinea pig gastric mucosa. Inhibiting the (Na⁺, K⁺) ATPase either by serosal addition of ouabain or by establishing K⁺-free mucosal and serosal conditions abolished net Cl^– transport. Depolarizing the cell membrane potential with triphenylmethylphosphonium (a lipid-soluble cation), and hence reducing both the Na⁺ and Cl^– electrochemical potential gradients, resulted in inhibition of net Cl^– flux. Reduction of short-circuit current on replacing Na⁺ by choline in the serosal bathing solution was shown to be due to inhibition of Cl^– transport. Serosal addition of diisothiocyanodisulfonic acid stilbene (an inhibitor of anion transport systems) abolished net Cl^– flux but not net Na⁺ flux. These results are compatible with the proposed model of a Cl^–/Na⁺ cotransport mechanism governing serosal Cl^– entry into the secreting cells. We suggest that the same mechanism may well facilitate both coupled Cl^–/Na⁺ entry and coupled HCO ₃ ^– /Na⁺ exit on the serosal side of the tissue.     

Measurement and stoichiometry of bumetanide-sensitive (2Na∶1K∶3Cl) cotransport in ferret red cells

Summary The bumetanide-sensitive uptake of Na⁺, K^–(Rb^–) and Cl^– has been measured at 21°C in ferrent red cells treated with (SITS+DIDS) to minimize anion flux via capnophorin (Band 3). During the time course of the influx experiments tracer uptake was a first-order rate process. At normal levels of external Na⁺ (150mm) the bumetanide-sensitive uptake of K⁺ was dependent on Cl^– and represented almost all of the K⁺ uptake, the residual flux demonstrating linear concentration dependence. The uptake of Na⁺ and Cl^– was only partially inhibited by bumetanide indicating that pathways other than (Na+K+Cl) cotransport participate in these fluxes. The diuretic-sensitive uptake of Na⁺ or Cl^– was, however, abolished by the removal of K⁺ or the complementary ion indicating that bumetanide-sensitive fluxes of Na⁺, K⁺ and Cl^– are closely coupled. At very low levels of [Na] _o (<5mm) K⁺ influx demonstrated complex kinetics, and there was evidence of the unmasking of a bumetanide-sensitive Na⁺-independent K⁺ transport pathway. The stoichiometry of bumetanide-sensitive tracer uptake was 2Na1K3Cl both in cells suspended in a low and a high K⁺-containing medium. The bumetanide-sensitive flux was markedly reduced by ATP depletion. We conclude that a bumetanide-sensitive cotransport of (2Na1K3Cl) occurs as an electroneutral complex across the ferret red cell membrane.     

Na+, K+, Cl− cotransport and its regulation in Ehrlich ascites Tumor cells. Ca2+/Calmodulin and protein kinase C dependent pathways

Summary Net Cl^– uptake as well as unidirectional³⁶Cl influx during regulatory volume increase (RVI) require external K⁺. Half-maximal rate of bumetanide-sensitive³⁶Cl uptake is attained at about 3.3mm external K⁺. The bumetanide-sensitive K⁺ influx found during RVI is strongly dependent on both Na⁺ and Cl^–. The bumetanide-sensitive unidirectional Na⁺ influx during RVI is dependent on K⁺ as well as on Cl^–. The cotransporter activated during RVI in Ehrlich cells, therefore, seems to transport Na⁺, K⁺ and Cl^–. In the presence of ouabain and Ba⁺ the stoichiometry of the bumetanide-sensitive net fluxes can be measured at 1.0 Na⁺, 0.8 K⁺, 2.0 Cl^– or approximately 1 : Na, 1 : K, 2 : Cl. Under these circumstances the K⁺ and Cl^– flux ratios (influx/efflux) for the bumetanide-sensitive component were estimated at 1.34 ±0.08 and 1.82 ± 0.15 which should be compared to the gradient for the Na⁺, K⁺, 2Cl^– cotransport system at 1.75 ± 0.24.Addition of sucrose to hypertonicity causes the Ehrlich cells to shrink with no signs of RVI, whereas shrinkage with hypertonic standard medium (all extracellular ion concentrations increased) results in a RVI response towards the original cell volume. Under both conditions a bumetanide-sensitive unidirectional K⁺ influx is activated. During hypotonic conditions a small bumetanide-sensitive K⁺ influx is observed, indicating that the cotransport system is already activated.The cotransport is activated 10–15 fold by bradykinin, an agonist which stimulates phospholipase C resulting in release of internal Ca²⁺ and activation of protein kinase C.The anti-calmodulin drug pimozide inhibits most of the bumetanide-sensitive K⁺ influx during RVI. The cotransporter can be activated by the phorbol ester TPA. These results indicate that the stimulation of the Na⁺, K⁺, Cl^– cotransport involves both Ca²⁺/calmodulin and protein kinase C.     

Kinetics and Energetics of Light-enhanced Potassium Absorption by Corn Leaf Tissue

The effect of illumination on the absorption of K⁺ by leaf tissue of Zea mays was investigated. The rate of K⁺ absorption was enhanced by exposure of slices of corn leaf tissue to light, even of relatively low intensities. Potassium was transported inward, with virtually no efflux of previously accumulated K⁺. The evidence indicates that the transport mechanism for absorption of K⁺ is the same in the light as in the dark, but that the source of energy for absorption of K⁺ is different in the light from that in the dark. Various anti-metabolites were used to establish that the energy utilized for active ion transport in the light came partly from ATP supplied by cyclic photophosphorylation. Expenditure of ATP was required in the dark too, but this ATP was formed by oxidative phosphorylation. Establishing the ultimate source of energy for active ion uptake by higher plants might be facilitated by demonstration of an ion-transport process that is not linked directly with the transfer of electrons in the mitochondrial cytochrome chain.     

The mechanism of Cl− transport at the plasma membrane ofChara corallina I. Cotransport with H+

Summary Cl^– transport into cells ofChara corallina was studied in relation to that of other ions which have been proposed as cosubstrates for the Cl^– transport system. Although there appears to be a partial mutual dependence between K⁺ and Cl^– for transport in intact cells, this is not apparent in cells which have been perfused internally. Moreover, in intact cells, the fluxes of K⁺ and Cl^– show a large degree of independence in their responses to Cl^– starvation. Cl^– transport is electrogenic in a direction indicating the transport of excess positive charge into the cell. In the absence of any other likely counter ion, it is suggested that Cl^– is cotransported with H⁺. Response of Cl^– influx to internal and external pH in perfused cells is consistent with this suggestion. There appears, in addition, to be a role for ATP in transport as judged by fourfold stimulation of Cl^– influx in perfused cells when 1mm ATP is incorporated in the perfusion medium.     

Separate,Ca2+-activated K+ and Cl− transport pathways in Ehrlich ascites tumor cells

Summary The net loss of KCl observed in Ehrlich ascites cells during regulatory volume decrease (RVD) following hypotonic exposure involves activation of separate conductive K⁺ and Cl^– transport pathways. RVD is accelerated when a parallel K⁺ transport pathway is provided by addition of gramicidin, indicating that the K⁺ conductance is rate limiting. Addition of ionophore A23187 plus Ca²⁺ also activates separate K⁺ and Cl^– transport pathways, resulting in a hyperpolarization of the cell membrane. A calculation shows that the K⁺ and Cl^– conductance is increased 14-and 10-fold, respectively. Gramicidin fails to accelerate the A23187-induced cell shrinkage, indicating that the Cl^– conductance is rate limiting. An A23187-induced activation of⁴²K and³⁶Cl tracer fluxes is directly demonstrated. RVD and the A23187-induced cell shrinkage both are: (i) inhibited by quinine which blocks the Ca²⁺-activated K⁺ channel. (ii) unaffected by substitution of NO ₃ ^– or SCN^– for Cl^–, and (iii) inhibited by the anti-calmodulin drug pimozide. When the K⁺ channel is blocked by quinine but bypassed by addition of gramicidin, the rate of cell shrinkage can be used to monitor the Cl^– conductance. The Cl^– conductance is increased about 60-fold during RVD. The volume-induced activation of the Cl^– transport pathway is transient, with inactivation within about 10 min. The activation induced by ionophore A23187 in Ca²⁺-free media (probably by release of Ca²⁺ from internal stores) is also transient, whereas the activation is persistent in Ca²⁺-containing media. In the latter case, addition of excess EGTA is followed by inactivation of the Cl^– transport pathway. These findings suggest that a transient increase in free cytosolic Ca²⁺ may account for the transient activation of the Cl^– transport pathway. The activated anion transport pathway is unselective, carrying both Cl^–, Br^–, NO ₃ ^– , and SCN^–. The anti-calmodulin drug pimozide blocks the volume- or A23187-induced Cl^– transport pathway and also blocks the activation of the K⁺ transport pathway. This is demonstrated directly by⁴²K flux experiments and indirectly in media where the dominating anion (SCN^–) has a high ground permeability. A comparison of the A23187-induced K⁺ conductance estimated from⁴²K flux measurements at high external K⁺, and from net K^– flux measurements suggests single-file behavior of the Ca²⁺-activated K⁺ channel. The number of Ca²⁺-activated K⁺ channels is estimated at about 100 per cell.     

Volume regulatory activity of the Ehrlich ascites tumor cell and its relationship to ion transport

Summary The volume regulatory response of the Ehrlich ascites tumor was studied in KCl-depleted, Na⁺-enriched cells. Subsequent incubation in K⁺-containing NaCl medium results in the reaccumulation of K⁺, Cl^–, water and the extrusion of Na⁺. The establishment of the physiological steady state is due primarily to the activity of 2 transport systems. One is the Na/K pump (K _M for K ₀ ⁺ =3.5mm;J _max=30.1 mEq/kg dry min), which in these experiments was coupled 1K⁺/1 Na⁺. The second is the Cl^–-dependent (Na⁺+K⁺) cotransport system (K _M for K ₀ ⁺ =6.8mm;J _max=20.8 mEq/kg dry min) which mediates, in addition to net ion uptake in the ratio of 1K⁺1Na⁺2Cl^–, the exchange of K _i ⁺ for K ₀ ⁺ . The net passive driving force on the cotransport system is initially inwardly directed but does not decrease to zero at the steady state. This raises the possibility of the involvement of an additional source of energy. Although cell volume increases concomitant with net ion uptake, this change does not appear to be a major factor regulating the activity of the cotransport system.     

Sidium-dependent ion cotransport in steady-state Ehrlich ascites tumor cells

Summary The Ehrlich tumor cell possesses and anion-cation cotransport system which operates as a bidirectional exchanger during the physiological steady state. This cotransport system, like that associated with the volume regulatory mechanism (i.e. coupled net uptake of Cl^–+Na⁺ and/or K⁺) is Cl^–-selective and furosemide-sensitive, suggesting the same mechanism operating in two different modes. Since Na⁺ has an important function in the volume regulatory response, its role in steady-state cotransport was investigated. In the absence of Na⁺, ouabain-insensitive K⁺ and DIDS-insensitive Cl^– transport (KCl cotransport) are low and equivalent to that found in 150mm Na⁺ medium containing furosemide. Increasing the [Na⁺] results in parallel increases in K⁺ and Cl^– transport. The maximum rate of each (18 to 20 meq/(kg dry wt)·min) is reached at about 20mm Na⁺ and is maintained up to 55mm. Thus, over the range 1 to 55mm Na⁺ the stoichiometry of KCl cotransport is 11. In contrast to K⁺ and Cl^–, furosemide-sensitive Na⁺ transport is undetectable until the [Na⁺] exceeds 50mm. From 50 to 150mm Na⁺, it progressively rises to 7 meq/(kg dry wt)·min, while K⁺ and Cl^– transport decrease to 9 and 16 meq/(kg dry wt)·min, respectively. Thus, at 150mm Na⁺ the stoichiometric relationship between Cl^–, Na⁺ and K⁺ is 211. These results are consistent with the proposal that the Cl^–-dependent cation cotransport system when operating during the steady state mediates the exchange of KCl for KCl or NaCl for NaCl; the relative proportion of each determined by the extracellular [Na⁺].     

Light-dependent potassium uptake by Pisum sativum leaf fragments

A net K⁺ influx into chopped pea leaves bathed in 5 mM KCl,0.26 M sucrose and illuminated with 4000 lux amounted to about7.5 µmoles/g fresh weight-hr, while essentially no netflux occurred in the dark. This light-dependent K⁺ uptake waslinear with time for nearly 2 hr and continuously increasedas the light intensity was raised to 9000 lux. Over half ofthe K⁺ uptake was balanced by H⁺ release into the bathing solution,possibly by a mechanism in which bicarbonate was the anion accompanyingK⁺. The replacement of Cl^– by HCO₃^– increased thelight-dependent K⁺ uptake to 56 µmoles/g fresh weight-hr.About 23% of the light-dependent K⁺ uptake in 5 mM KCl was accompaniedby a Cl^– uptake. This net Cl^– influx was less sensitiveto the uncoupler tri-Fl-CCP and more sensitive to DCMU in thebathing solution than was the K⁺ uptake. The remaining net K⁺influx into pea leaf fragments was balanced by effluxes of sodium(accounting for 5%), magnesium (8%) and calcium (1%). (Received March 31, 1969; )     

Reconstituted Cl− pump protein: A novel ion(Cl−)-motive ATPase

Cl^– absorption by theAplysia californica foregut is effected through an active Cl^– transport mechanism located in the basolateral membrane of the epithelial absorptive cells. These basolateral membranes contain both Cl^–-stimulated ATPase and ATP-dependent Cl^– transport activities which can be incorporated into liposomes via reconstitution. Utilizing the proteoliposomal preparation, it was demonstrated that ATP, and its subsequent hydrolysis, Mg²⁺, Cl^–, and a pH optimum of 7.8 were required to generate maximal intraliposomal Cl^– accumulation, electrical negativity, and ATPase activity. Additionally, an inwardly-directed valinomycininduced K⁺ diffusion potential, making the liposome interior electrically positive, enhanced both ATP-driven Cl^– accumulation and electrical potential while an outwardly-directed valinomycininduced K⁺ diffusion potential, making the liposome interior electrically negative, decreased both ATP-driven Cl^– accumulation and electrical potential compared with proteoliposomes lacking the ionophore. Either orthovanadate orp-chloromercurobenzene sulfonate inhibited both the ATP-dependent intraliposomal Cl^– accumulation, intraliposomal negative potential difference, and also Cl^–-stimulated ATPase activity. Both aspects of Cl^– pump transport kinetics and its associated catalytic component kinetics were the first obtained utilizing a reconstituted transporter protein. These results strongly support the hypothesis that Cl^–-ATPase actively transports Cl^– by an electrogenic process.     

Pivotal Role of Reduced Glutathione in Oxygen-induced Regulation of the Na<Superscript><Emphasis Type="Bold">+</Emphasis></Superscript>/K<Superscript><Emphasis Type="Bold">+</Emphasis></Superscript> Pump in Mouse Erythrocyte Membranes

This study addresses the mechanisms of oxygen-induced regulation of ion transport pathways in mouse erythrocyte, specifically focusing on the role of cellular redox state and ATP levels. Mouse erythrocytes possess Na⁺/K⁺ pump, K⁺-Cl^– and Na⁺-K⁺-2Cl^– cotransporters that have been shown to be potential targets of oxygen. The activity of neither cotransporter changed in response to hypoxia-reoxygenation. In contrast, the Na⁺/K⁺ pump responded to hypoxic treatment with reversible inhibition. Hypoxia-induced inhibition was abolished in Na⁺-loaded cells, revealing no effect of O₂ on the maximal operation rate of the pump. Notably, the inhibitory effect of hypoxia was not followed by changes in cellular ATP levels. Hypoxic exposure did, however, lead to a rapid increase in cellular glutathione (GSH) levels. Decreasing GSH to normoxic levels under hypoxic conditions abolished hypoxia-induced inhibition of the pump. Furthermore, GSH added to the incubation medium was able to mimic hypoxia-induced inhibition. Taken together these data suggest a pivotal role of intracellular GSH in oxygen-induced modulation of the Na⁺/K⁺ pump activity.